Association of obesity with leukocyte count in obese individuals without metabolic syndrome

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Original Article

Association of obesity with leukocyte count in obese individuals without metabolic syndrome Elena Ryder a,*, Marı´a Diez-Ewald a, Jesu´s Mosquera a, Erika Ferna´ndez a, ˜ ez b, Renata Vargas a, Caterina Pen ˜ a c, Nelson Ferna´ndez a Adriana Pedrean a b c

Instituto de Investigaciones Clı´nicas ‘‘Dr. Ame´rico Negrette’’, Facultad de Medicina, Universidad del Zulia, Maracaibo, Venezuela Ca´tedra de Inmunologı´a, Escuela de Bionanalisis, Facultad de Medicina, Universidad del Zulia, Maracaibo, Venezuela Ca´tedra de Gene´tica, Escuela de Bionanalisis, Facultad de Medicina, Universidad del Zulia, Maracaibo, Venezuela

A R T I C L E I N F O

A B S T R A C T

Keywords: Obesity Insulin resistance Leukocytes Inflammation

Aims: Inflammation in obesity is associated to insulin resistance (IR), hyperglycemia, hypertension and hyperlipidemia. Leukocytes play an important role in obesity associated inflammation. The initial factors that generate the inflammatory events in the obesity remain unclear. Therefore, the aim of this study was to determine the association of circulating leukocytes with clinical and biochemical parameters in obese individuals with clinical and biochemical parameters in normal range and with or without IR. Methods: Nineteen obese non-diabetic and 9 lean subjects were studied for serum levels of insulin, lipids, glycated hemoglobin, glycemia, for clinical parameters as HOMA-IR, arterial pressure and anthropometric parameters, and for leukocyte counts. Neutrophil/lymphocyte ratio (N/L) was calculated using the loge of leukocyte counts. Association between leukocytes and studied parameters was determined by Pearson’s correlation. Results: Two groups of obese individuals were observed: with high levels of insulin (with IR) and with normal levels (without IR). Positive correlations were observed between leukocyte and lymphocyte counts with body mass index and HOMA-IR and negative correlation with decreased HDL levels. Lymphocytes correlated with increased levels of insulin. Leukocytes and neutrophils correlated positively with increased visceral fat and liver steatosis. These associations were absent in the obese group without IR. N/L ratio did not show correlations with studied parameters. The leukocyte associations were mainly observed in obese individuals with IR. Conclusions: These data may represent initial leukocyte associations with morbidity features and define two different obese individuals that may evolve to the chronic inflammation observed in the obesity. ß 2014 Diabetes India. Published by Elsevier Ltd. All rights reserved.

1. Introduction Obesity is associated with pathologies that define the metabolic syndrome (MS). These include insulin resistance, hyperinsulinemia, impaired glucose tolerance, dyslipidemia, hypertension and obesity, in particular central adiposity [1]. The concurrence of any three of these conditions will result in a diagnosis of MS. Insulin resistance (IR) is a complication of chronic inflammation associated with obesity that increases the risk of developing metabolic diseases, resulting from the alteration of insulin-mediated signaling pathway

* Corresponding author at: Apartado Postal 23, Maracaibo, 4001-A, Zulia, Venezuela. Tel.: +58 261 7114752; fax: +58 261 7916053. E-mail address: [email protected] (E. Ryder).

leading to hyperglycemia and other alterations such as hypertension and hyperlipidemia [1]. This chronic inflammation has been focused on monocyte/macrophage infiltration and activation in the adipose tissue. However, unique roles have been shown for a variety of immune cells in this tissue, including cells from both innate and adaptive immune system [2–5]. Circulating leukocytes represent a key factor in the study of the pathogenesis of obesity. In this regard, association between leukocyte count and diabetes risk has been recently suggested [6,7]. It has been clearly demonstrated that the inflammatory status is a causative factor in diet-induced insulin resistance, but it remains unclear what is/are the initial(s) association of leukocytes and leukocyte subtypes with morbidity factors involved in MS. The obese individuals without clinical and biochemical alterations could represent an initial stage of inflammatory status during the

http://dx.doi.org/10.1016/j.dsx.2014.09.002 1871-4021/ß 2014 Diabetes India. Published by Elsevier Ltd. All rights reserved.

Please cite this article in press as: Ryder E, et al. Association of obesity with leukocyte count in obese individuals without metabolic syndrome. Diab Met Syndr: Clin Res Rev (2014), http://dx.doi.org/10.1016/j.dsx.2014.09.002

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obesity. Therefore, the aim of this study was to evaluate the associations of clinical and biochemical parameters with circulating leukocyte and leukocyte subtype counts in obese individuals with normal clinical and biochemical parameters and with or without IR. 2. Subjects Forty-four obese individuals were analyzed and chosen through a voluntary call to participate in the project. Only 19 obese nondiabetic subjects (20–55 years of age), with body mass index (BMI) greater than 30 kg/m2 and 9 lean control individuals, whose BMI was less than 25 kg/m2, were selected in this study. All subjects fulfilled the inclusion criteria: absence of arterial hypertension, diabetes or other metabolic abnormalities, not evidence of hepatitis or HIV infection (determined by the absence of antibodies against the viruses), absence of any other current infectious processes, not taken any medications known to influence glucose or lipid metabolism or the inflammatory pathway, and in the case of women, not under hormonal contraceptive drugs. The project was approved by the Ethic Committee of the Instituto de Investigaciones Clı´nicas ‘‘Dr. Ame´rico Negrette’’, University of Zulia in Maracaibo, Venezuela, according with the principles of the Declaration of Helsinki as revised in 2008. Written consent was obtained from all subjects. Individual demographic information was collected including age, sex, qualifying criteria, current medications, height, weight and waist circumference. Body mass index was calculated from the formula: weight (kg)/height (m2), considering obesity when it was >30 kg/m2. Absence of hypertension was confirmed by means of a sphyngomanometer. Ultrasound was used for the estimation of visceral fat and hepatic steatosis as described by Ryder et al. [8]. Grades of hepatic steatosis were assigned arbitrarely for statistical purposes; 1 represents: I/III, 2: I–II/III, 3: II/III and 4: II–III/III. 3. Materials and methods 3.1. Biochemical and hematological measurements To each subject a fasting blood sample was withdrawn for the determination of biochemical parameters. Glucose, total cholesterol and triglycerides were measured enzymatically; high density

lipoprotein (HDL)-cholesterol was measured after precipitation of the apo B-containing lipoproteins (Human GmbH, Germany). Plasma insulin was determined by chemiluminescence immunoassay (IMMULITE 1000, Siemens Diagnostics, USA) and total glycated hemoglobin by the method of Bioscience Medical SL, Spain. Homeostasis model assessment of insulin resistance (HOMA-IR) was calculated as: fasting insulin (IU/L)  fasting glucose (mmol/L)/22.5 according to Mathews et al. [9], and the cut off point to consider IR was 2.6 [10,11]. The number of leukocytes and leukocyte subtypes were computed with an autoanalyzer (Beckman Coulter Counter, Coulters Corporation, FL, USA). The neutrophil: lymphocyte ratio was defined as the loge neutrophil count/loge lymphocyte count. 3.2. Statistical analysis The data were normally distributed and are expressed as the mean  standard error of the mean. Significant differences among groups were analyzed by ANOVA followed by Dunnet’s test for multiple comparisons. Pearson’s correlations were estimated for various blood cell counts with the numbers of obesity, metabolic and clinical markers using PRISM statistical software (GraphPad Software). Differences were considered statistically significant at p < 0.05. 4. Results None of the lean and obese individuals in this study had hypertension, hyperglycemia or elevated glycated hemoglobin, and their mean values for total and LDL cholesterol were similar among the three groups. As expected, the BMI, HOMA-IR, waist circumference and visceral fat of the obese with IR and obese without IR were elevated in relation to the control group; however, the obese with IR showed significantly increased values of BMI, HOMA-IR and insulin compared to the obese without IR. Triglyceride values were higher and HDL cholesterol lower in both obese groups; however, the mean values were in the normal range for this population (Table 1). Increased total leukocyte, neutrophil and lymphocyte counts were observed in obese individuals with IR (Fig. 1); however, the total values remained into the normal range. Positive correlations were observed between total leukocyte and lymphocyte counts with BMI and

Table 1 Clinical and laboratory parameters in obese individuals with or without insulin resistance and in healthy lean control individuals. Parameter

N Age (yrs) Body mass index (kg/m2) Systolic blood pressure (mmHg) Diastolic blood pressure (mmHg) Homa-IR Waist circumference (cm) Insulin (mU/mL) Glycemia (mg/dL) Glycated hemoglobin (%) Triglycerides (mg/dL) Cholesterol (mg/dL)

Control (A)

Ob IR (B)

A vs. B

A vs. C

B vs. C

p value

p value

p value

NS

NS

NS

<0.01 <0.05 <0.01

<0.01 NS NS

<0.05 NS <0.01

<0.01

NS

<0.01

<0.01

<0.01

NS

<0.01 <0.05 NS

NS NS NS

<0.01 NS NS

127.9  13.2

<0.05

<0.05

NS

187.1  14.4

NS

NS

NS

<0.05

<0.05

NS

NS

NS

NS

<0.01

<0.01

NS

9 29.7  3.3

9 33.3  4.2

10 38.7  3.4

22.4  0.5 112.5  2.5 75  1.9

37.8  0.8 122.2  2.8 85.1  1.7

34.6  1.2 114.0  2.7 76  1.6

0.83  0.2 77  2.0 4.1  1.0 80.3  3.8 6.11  0.14 80.3  3.8

5.83  0.7 114.9  4.2 25.3  2.8 92.8  3.4 6.37  0.36 126.6  14.7

163.9  7.9

186  14.0

HDL-cholesterol (mg/dL)

51.8  2.5

41  2.4

LDL-cholesterol (mg/dL)

99.8  6.2

Visceral fat (cm)

Ob no IR (C)

2.36  0.3

119.5  11.0 6.2  0.6

1.48  0.2 106  2.4 6.93  0.9 86.4  1.6 6.44  0.14

42  3.3 117.4  14.5 5.4  0.4

Ob IR: obese with insulin resistance; Ob no IR: obese without insulin resistance; values are expressed as mean  standard error.

Please cite this article in press as: Ryder E, et al. Association of obesity with leukocyte count in obese individuals without metabolic syndrome. Diab Met Syndr: Clin Res Rev (2014), http://dx.doi.org/10.1016/j.dsx.2014.09.002

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Fig. 1. Leukocyte counts in controls, obese individuals with insulin resistance (IR) and without insulin resistance (no IR). Significant increased counts were observed in total leukocytes (A), neutrophils (B) and lymphocytes (C). Values of monocytes (C) remained similar in the studied groups.

Table 2 Correlations between different leukocyte types and clinical and biochemical parameters in obese individuals with insulin resistance. Parameters

Total leukocytes (103 mL)

Total neutrophils (103 mL)

Total monocytes (103 mL)

Total lymphocytes (103 mL)

Age (years)

r = 0.2541 p = 0.3089 r = 0.4791 p = 0.0442 r = 0.4960 p = 0.0363 r = 0.4471 p = 0.0629 r = 0.4680 p = 0.0502 r = 0.7173 p = 0.0086 r = 0.9202 p = 0.0033 r = 0.9084 p < 0.0001 r = 0.6819 p = 0.0018 r = 0.5902 p = 0.0099 r = 0.1178 p = 0.6415 r = 0.1761 p = 0.4846 r = S0.5694 p = 0.0137 r = 0.05931 p = 0.8152 r = 0.2529 p = 0.3114 r = 0.2860 p = 0.2499

r = 0.1274 p = 0.6145 r = 0.3556 p = 0.1476 r = 0.3146 p = 0.2036 r = 0.2944 p = 0.2356 r = 0.3618 p = 0.1401 r = 0.7224 p = 0.008 r = 0.5966 p = 0.04 NA

r = 0.07535 p = 0.7663 r = 0.3268 p = 0.1856 r = 0.3597 p = 0.1426 r = 0.3099 p = 0.2108 r = 0.2128

r = 0.3992 p = 0.1007 r = 0.4755 p = 0.046 r = 0.5792 p = 0.011 r = 0.5037 p = 0.033 r = 0.4592 p = 0.0552 r = 0.3360 p = 0.2856 r = 0.5122 p = 0.0887 r = 0.3239 p = 0.1898 NA

Body Mass Index (kg/m2) HOMA-IR Insulin (mU/mL) Glucose (mg/dL) Visceral Fat (cm) Hepatic steatosis (grades) Neutrophils (103 mL) Lymphocytes (103 mL) 3

Monocytes (10 mL) Triglycerides (mg/dL) Cholesterol (mg/dL) HDL (mg/dL) LDL (mg/dL) Systolic blood pressure (mmHg) Diastolic blood pressure (mmHg)

NA

r = 0.2721 p = 0.3923 r = 0.2379 p = 0.4564 r = 0.3710 p = 0.1295 r = 0.5556 p = 0.0167 NA

r = 0.006033 p = 0.9810 r = 0.2611 p = 0.2954 r = 0.4546 p = 0.0581 r = 0.1573 p = 0.5330 r = 0.1384 p = 0.5838 r = 0.2872 p = 0.2479

r = 0.07493 p = 0.7676 r = 0.1634 p = 0.5170 r = 0.2033 p = 0.4185 r = 0.1539 p = 0.5421 r = 0.2316 p = 0.3552 r = 0.2231 p = 0.3735

NA

NA r = 0.3122 p = 0.2072 r = 0.09150 p = 0.7180 r = S0.5428 p = 0.0199 r = 0.1863 p = 0.4592 r = 0.3362 p = 0.1725 r = 0.1421 p = 0.1421

NA: no apply.

Please cite this article in press as: Ryder E, et al. Association of obesity with leukocyte count in obese individuals without metabolic syndrome. Diab Met Syndr: Clin Res Rev (2014), http://dx.doi.org/10.1016/j.dsx.2014.09.002

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HOMA-IR. These cells were also negatively correlated with HDLcholesterolemia. Lymphocytes were also correlated with increased serum insulin. Total leukocytes and neutrophils were correlated with visceral fat and hepatic steatosis. Leukocytes were correlated with neutrophil, lymphocyte and monocyte counts and lymphocytes with monocyte counts (Table 2 and Figs. 2 and 3). Obese individuals without IR only showed correlations between total leukocytes and all leukocyte subtype counts and neutrophils with monocyte counts (Table 3, Fig. 4). There were no statistical significances when the neutrophil:lymphocyte ratio was correlated with the studied groups (Fig. 5), or with the antropometric, clinical or biochemical parameters. 5. Discussion Obesity is a major risk factor for cardio-metabolic morbidity. Current approaches relate comorbidities such as dyslipidemia, hyperglycemia, hypertension and alterations in anthropometric or imaging-based for adipose tissue distribution parameters with obesity. In addition, pro-inflammatory events have been shown to

correlate in humans with adipose tissue inflammation and with obesity-associated health risks [12]. In this study two groups of obese subjects were analyzed. Morbidity alterations such as dyslipidemia, hyperglycemia and hypertension were absent suggesting lack of cardio-metabolic morbidity and probably an initial stage of morbidity alterations. Leucocytes can infiltrate adipose tissue in obesity and produce cytokines and other products capable of inducing inflammation [12,13]. The association of leukocytes with biochemical and clinical parameters during MS and diabetes have been documented. In this regard, previous analyses showed correlation between HOMA IR, insulin and obesity parameters in individuals with the presence of one or more risk factors for type 2 diabetes, suggesting that hematological variables including leukocyte count may be related to the underlying pathophysiological changes associated with type 2 diabetes mellitus [14]. Meta-analysis studies have shown that an increased count of circulating leukocytes is associated with higher risk of type 2 diabetes [15]. In addition, association of all subtypes of circulating leukocytes with IR have been shown in high risk individuals [16]. In this study, obese individuals with increased

Fig. 2. Correlation of leukocyte counts with anthropometric and metabolic variables. Lymphocytes were positively correlated with body mass index (A) BMI, HOMA-IR (B) and insulin (C) and negatively correlated with decreased levels of HDL (D). Neutrophils were positively correlated with visceral fat (E) and hepatic steatosis (G). Lean and obese individuals with IR were included in these analyses. Correlation coefficients were determined by Pearson’s correlation analysis.

Please cite this article in press as: Ryder E, et al. Association of obesity with leukocyte count in obese individuals without metabolic syndrome. Diab Met Syndr: Clin Res Rev (2014), http://dx.doi.org/10.1016/j.dsx.2014.09.002

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Fig. 3. Correlation of leukocyte counts with leukocyte subtypes. Total leukocyte counts were positively correlated with neutrophil (A), lymphocyte (B) and monocyte (C) counts. Lymphocytes were positively correlated with monocyte counts (D). Lean and obese individuals with IR were included in these analyses. Correlation coefficients were determined by Pearson’s correlation analysis.

Table 3 Correlations between different leukocyte types and clinical and biochemical parameters in obese individuals without insulin resistance. Parameter Age (years) Body Mass Index (kg/m2) HOMA IR Insulin (mU/mL) Glucose (mg/dL) Visceral Fat (cm) Hepatic steatosis (grades) Neutrophils (103 mL) Lymphocytes (103 mL) Monocytes (103 mL) Triglycerides (mg/dL) Cholesterol (mg/dL) HDL (mg/dL) LDL (mg/dL) Systolic blood pressure (mmHg) Diastolic blood pressure (mmHg)

Total leukocytes (103 mL)

Total neutrophils (103 mL)

Total monocytes (103 mL)

Total lymphocytes (103 mL)

r = 0.2541 p = 0.3089 r = 0.05279 p = 0.8405 r = 0.2296 p = 0.3755 r = 0.2216 p = 0.3928 r = 0.006713 p = 0.9796 r = 0.1998 p = 0.5558 r = 0.2859 p = 0.4232 r = 0.8983 p < 0.0001 r = 0.6485 p = 0.0049 r = 0.5584 p = 0.0198 r = 0.06284 p = 0.8106 r = 0.004831 p = 0.9853 r = 0.2968 p = 0.2473 r = 0.04897 p = 0.8519 r = 0.2783 p = 0.2795 r = 0.05825 p = 0.8243

r = 0.1274 p = 0.6145 r = 0.1958 p = 0.4362 r = 0.1459 p = 0.5763 r = 0.1178 p = 0.6525 r = 0.1124 p = 0.6677 r = 0.2673 p = 0.4268 r = 0.3272 p = 0.3561 NA NA NA NA NA NA r = 0.02045 p = 0.9379 r = 0.02873 p = 0.9129 r = 0.1446 p = 0.5798 r = 0.07056 p = 0.7879 r = 0.3038 p = 0.2358 r = 0.06439 p = 0.8060

r = 0.07535 p = 0.7663 r = 0.3268 p = 0.1856 r = 0.2657 p = 0.3028 r = 0.06087 p = 0.8165 r = 0.2657 p = 0.3028 r = 0.2462 p = 0.4655 r = 0.2379 p = 0.4564 r = 0.5006 p = 0.0407 r = 0.2243 p = 0.3868 NA NA r = 0.04089 p = 0.8762 r = 0.3270 p = 0.2002 r = 0.1026 p = 0.6951 r = 0.3745 p = 0.1387 r = 0.2094 p = 0.4200 r = 0.2030 p = 0.4345

r = 0.3992 p = 0.1007 r = 0.3121 p = 0.2226 r = 0.1445 p = 0.5800 r = 0.1310 p = 0.6044 r = 0.08396 p = 0.7572 r = 0.5551 p = 0.0763 r = 0.07516 p = 0.8261 r = 0.2089 p = 0.4055 NA NA NA NA r = 0.3751 p = 0.1379 r = 0.05498 p = 0.8340 r = 0.3753 p = 0.1377 r = 0.01987 p = 0.9397 r = 0.04385 p = 0.8673 r = 0.2557 p = 0.3219

NA: no apply.

Please cite this article in press as: Ryder E, et al. Association of obesity with leukocyte count in obese individuals without metabolic syndrome. Diab Met Syndr: Clin Res Rev (2014), http://dx.doi.org/10.1016/j.dsx.2014.09.002

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Fig. 4. Correlation of leukocyte counts with leukocyte subtypes. Total leukocyte counts were positively correlated with neutrophil (A), lymphocyte (B) and monocyte (C) counts. Neutrophils were positively correlated with monocyte counts (D). Lean and obese individuals without IR were included in these analyses. Correlation coefficients were determined by Pearson’s correlation analysis.

concentration of serum insulin showed higher numbers of total leukocytes, neutrophils and lymphocytes suggesting inflammatory response. Leukocytes and lymphocytes correlated with anthropometric parameters of obesity and serum HDL concentration; lymphocytes correlated with increased levels of insulin, suggesting a role of these cells in the induction of risk factors for MS during early stage of obesity, when comorbidities are still not present. Therefore, our findings suggest that lymphocytes in the periods of absence of risk factors, could be the key cells in the initiation of obesity morbidity. Similar findings have been documented in diabetes. Increased total leukocytes, neutrophil and lymphocyte counts were detected in individuals with increased risk of diabetes; the stronger association was observed in lymphocytes, suggesting that this association was related to insulin sensitivity rather than subclinical inflammation [7]. Since our findings were presented only in obese individuals with IR, a common pathogenic mechanism in obesity and diabetes could be suggested. However, other report showed that elevated leukocyte counts due to increased number of neutrophil in

Fig. 5. Neutrophil/lymphocyte ratio (N/L) in controls, obese individuals with insulin resistance (IR) and without insulin resistance (no IR). N/L was observed similar in all studied groups.

nascent MS could contribute to the increased risk for both, diabetes and cardiovascular disease [17]. Insulin resistance during obesity is secondary to an inflammatory response caused by the infiltration of adipose tissue by monocytes and other circulating leukocytes [2–5], resulting in altered insulin-mediated signaling pathway and in hyperglycemia. IR is linked with other alterations such as hypertension and hyperlipidemia [1,18,19]. Monocyte/macrophages are the predominant leukocyte population in fat and contribute to obesityinduced inflammation [20]. In the present study, the patients with high levels of insulin, and clinical and metabolic parameters in the normal range, and a HOMA-IR > 2.6, did not show the expected consequences related to IR, suggesting a sustained homeostasis or an early presence of IR status. In addition, increased lymphocyte and neutrophil counts and their correlations with risk factors were only observed in this obese group. Increased number of monocyte/ macrophages an correlations with risk factors were absent, suggesting that in this period of obesity, lymphocytes and neutrophils play a more important role than monocytes. In this regard, T and B lymphocytes and neutrophils have been reported to be involved in IR during obesity [21,22]. Previous studies have shown an association between leukocyte subtype counts and dyslipidemia in apparently healthy individuals, obesity, MS and diabetes type 2 [23–25]. Obese individuals in this study can not be classified as having MS or be diabetic individuals; however, significant negatively correlations were observed between total leukocyte and lymphocyte counts and hypo-HDL cholesterolemia, suggesting a role of lymphocytes in the decreased levels of serum HDL. Since, lymphocytes were also positively correlated with BMI, HOMA-IR and insulin levels, these initial associations could be increased in advanced progression of obesity with MS and diabetes type 2 and involve other leukocyte subtypes. In this regard, total leukocyte, neutrophil, lymphocyte, monocyte and eosinophil counts have been found higher in the

Please cite this article in press as: Ryder E, et al. Association of obesity with leukocyte count in obese individuals without metabolic syndrome. Diab Met Syndr: Clin Res Rev (2014), http://dx.doi.org/10.1016/j.dsx.2014.09.002

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patients with MS and leukocyte count was positively correlated with systolic and diastolic blood pressures, triglyceride, insulin, and HOMA-IR and negatively with HDL cholesterol in type 2 diabetic patients [24]. The association of lymphocytes with increased insulin and decreased serum HDL could induce deleterious effects on lymphocytes. It has been reported that increased susceptibility of lymphocytes to apoptosis correlated with a higher risk of insulin resistance and lipid disturbance in overweight and obese children [25]. As an unexplained finding, increased counts of total leukocytes and leukocyte subtypes were found in obese individuals with high levels of serum insulin (IR), suggesting the presence of low-grade systemic inflammation, a known pathogenic mechanism underlying most long-term complications of obesity. However, previous report from our group revealed that analyses of several inflammatory markers in these subjects were absent. In contrast, obese individuals without IR showed increased expression of inflammatory markers, suggesting an anti-inflammatory effect of insulin in the obese group with higher values of serum insulin [26]. In this study neutrophil counts from patients with IR were correlated with increased visceral fat and with hepatic steatosis parameters. Visceral fat is more significantly correlated with inflammation markers and oxidative stress than is subcutaneous fat. Morbid obesity is commonly associated with adipose tissue inflammation, which is characterized by an increased expression of various pro-inflammatory cytokines and cellular infiltrate including monocytes/macrophages, neutrophils, B lymphocytes, T lymphocytes, and others [27]. Relationship between neutrophils and circulating lipids in humans have been documented; after a oral fat load, postprandial triglyceride increments parallel with a neutrophil increment [28]. In human obesity neutrophil gelatinase-associated lipocalin in visceral adipose tissue is related to potential roles in insulin resistance and inflammation [29]. In addition, circulating activated neutrophils has been associated with morbid obesity, suggesting continuous activation of the innate immune system [30]. The correlation between increased visceral adipose tissue with increased circulating neutrophils in this study, suggests a contribution of neutrophil in the increase of visceral fat. Accordingly, increased adiposity associated to higher circulating neutrophil counts, suggesting acute inflammation have been reported in obese adolescents [31]. Neutrophils are the most abundant circulating white blood cell type in humans, and threir inappropriate activation and homing to the microvasculature, contribute to the pathological manifestations of many types of liver disease [32]. Nonalcoholic steatohepatitis (NASH) is a chronic inflammatory liver disease associated with IR and its metabolic consequences. Previously it has been shown in liver biopsies of severe obese subjects with NASH an association and correlation with hepatic neutrophil sequestration [33]. As shown in a previous report, these studied obese individuals with increased plasmatic levels of insulin and without IR metabolic consecuences, cannot be classified as morbid or severe obesity [26]. In addition, neutrophils were not correlated with IR and correlations were only restricted to tissue lipid deposits, suggesting an initial neutrophil infiltration in the liver and adipose tissue that can involve further inflammatory processes. The neutrophil/lymphocyte ratio (N/L), a new marker for predicting steatohepatitis and fibrosis in patients with nonalcoholic fatty liver disease is higher in these types of patients [34]. In this study, no significant differences were found when N/L values were correlated with the studied groups or with the different parameters, suggesting that N/F is not a useful marker in obesity with few morbidity factors. In conclusion, this study showed obese individuals without abnormal biochemical and clinical parameters, in which, one group with elevated values of serum insulin, showed leukocyte

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correlations with morbidity parameters of MS, and the other group with normal levels of insulin, did not. These obese individuals may represent two different aspects of initial stages of the chronic inflammation found in the obesity. Further studies are required to determine the evolution of obese individuals with high insulin levels, but, with normal clinical and biochemical parameters and the presence of inflammatory events over time. Source of funding This manuscript was supported by Consejo de Desarrollo Cientı´fico y Humanı´stico de la Universidad del Zulia (CC-0440-10, Maracaibo, Venezuela). Acknowledgements We thank Lissette Cornell, Nora Palazzi and Volga Mijac for excellent laboratory and clinical assistance (Instituto de Investigaciones Clı´nicas Dr. Ame´rico Negrette’’, Facultad de Medicina, Universidad del Zulia, Maracaibo, Venezuela). Conflict of interests: The authors declare no conflicts of interest. References [1] Cornier MA, Dabelea D, Hernandez TL, Lindstrom RC, Steig AJ, Stob NR, et al. The metabolic syndrome. Endocr Rev 2008;29(7):777–822. http://dx.doi.org/ 10.1210/er.2008-0024. [2] Reaven GM. The insulin resistance syndrome: definition and dietary approaches to treatment. Annu Rev Nutr 2005;25(4):391–406. [3] Osborn O, Olefsky JM. The cellular and signaling networks linking the immune system and metabolism in disease. Nat Med 2012;18(3):363–74. http:// dx.doi.org/10.1038/nm.2627. [4] Romeo GR, Lee J, Shoelson SE. Metabolic syndrome, insulin resistance, and roles of inflammation – mechanisms and therapeutic targets. Arterioscler Thromb Vasc Biol 2012;32(8):1771–6. http://dx.doi.org/10.1161/ATVBAHA.111.241869. [5] Watanabe Y, Nagai Y, Takatsu K. Activation and regulation of the pattern recognition receptors in obesity-induced adipose tissue inflammation and insulin resistance. Nutrients 2013;5(9):3757–78. http://dx.doi.org/10.3390/ nu5093757. [6] Twig G, Afek A, Shamiss A, Derazne E, Tzur D, Gordon B, et al. White blood cells count and incidence of type 2 diabetes in young men. Diabetes Care 2013;36(2):276–82. http://dx.doi.org/10.2337/dc11-2298. [7] Lorenzo C, Hanley AJ, Haffner SM. Differential white cell count and incident type 2 diabetes: the Insulin Resistance Atherosclerosis Study. Diabetologı´a 2014;57:83–92. http://dx.doi.org/10.1007/s00125-013-3080-0. [8] Ryder E, Mijac V, Ferna´ndez E, Palazzi N, Morales MC, Connell L, et al. Esteatosis hepa´tica, grasa visceral y alteraciones metabo´licas en individuos con sobrepeso/obesidad aparentemente sanos. Invest Clin 2014;55:3–14. [9] Matthews DR, Hosker JP, Rudenski AS, Naylor BA, Treacher DF, Turner RC. Homeostasis model assessment: insulin resistance and beta-cell function from fasting plasma glucose and insulin concentrations in man. Diabetologia 1985;28(7):412–9. [10] Buccini G, Wolftbal DL. Valores de corte para indices de insulinoresistencia, insulinosensibilidad e insulinosecrecion derivados de la formula HOMA y del programa HOMAZ. Interpretacio´n de los datos. Rev Argent Endocrinol Metab 2008;45:3–21. [11] Garmedia ML, Lera L, Sanchez H, Vauy R, Albala C. Homeostasis model assessment (HOMA) values in Chilean elderly subjects. Rev Med Chile 2009;137(11):1409–16. /S0034-98872009001100001. [12] Pecht T, Gutman-Tirosh A, Bashan N, Rudich A. Peripheral blood leucocyte subclasses as potential biomarkers of adipose tissue inflammation and obesitysubphenotypes in humans. Obes Rev 2014;15(4):322–37. http:// dx.doi.org/10.1111/obr.12133. [13] Carvalheira JB, Qiu Y, Chawla A. Blood spotlight on leukocytes and obesity. Blood 2013;122(19):3263–7. http://dx.doi.org/10.1182/blood-2013-04459446. [14] Hanley AJ, Retnakaran R, Qi Y, Gerstein HC, Perkins B, Raboud J, et al. Association of hematological parameters with insulin resistance and beta-cell dysfunction in nondiabetic subjects. Clin Endocrinol Metab 2009;94(10):3824– 32. http://dx.doi.org/10.1210/jc.2009-0719. [15] Gkrania-Klotsas E, Ye Z, Cooper AJ, Sharp SJ, Luben R, Biggs ML, et al. Differential white blood cell count and type 2 diabetes: systematic review and metaanalysis of cross-sectional and prospective studies. PLoS ONE 2010;5(10):e13405. http://dx.doi.org/10.1371/journal.pone.0013405. [16] Lee CT, Harris SB, Retnakaran R, Gerstein HC, Perkins BA, Zinman B, et al. White blood cell subtypes, insulin resistance and b-cell dysfunction in high-risk individuals – the PROMISE cohort. Clin Endocrinol (Oxf) 2013. http:// dx.doi.org/10.1111/cen.12390.

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Please cite this article in press as: Ryder E, et al. Association of obesity with leukocyte count in obese individuals without metabolic syndrome. Diab Met Syndr: Clin Res Rev (2014), http://dx.doi.org/10.1016/j.dsx.2014.09.002